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Abstract:

A method for diagnosing a genetic predisposition in a subject for
diseases, disorders or conditions including a diabetic kidney
complication such as kidney disease of type 2 diabetes or type 1
diabetes, end stage renal disease (ESRD) due to type 2 diabetes, ESRD due
to hypertension in type 2 diabetes, ESRD due to type 1 diabetes;
cardiovascular diseases due to type 2 diabetes or type 1 diabetes such as
atherosclerotic peripheral vascular disease, hypertension, ischemic
cardiomyopathy, and myocardial infarction due to type 2 diabetes or type
1 diabetes; and cerebrovascular accident due to type 2 diabetes. At least
one polynucleotide is analyzed to detect a single nucleotide polymorphism
(SNP), in which the presence of the single nucleotide polymorphism
indicates that the subject is suffering from, at risk for, or suspected
of suffering from the diseases, disorders or conditions. Also provided is
an array or kit for diagnosing the genetic predisposition.

Claims:

1. A method for diagnosing a genetic predisposition in a subject for a
disease, disorder or condition comprising a diabetic kidney complication
selected from the group consisting of end stage renal disease (ESRD) due
to type 2 diabetes, ESRD due to hypertension in type 2 diabetes, ESRD due
to type 1 diabetes; kidney disease of type 2 diabetes or type 1 diabetes;
cardiovascular disease due to type 2 diabetes or type 1 diabetes; and
cerebrovascular accident due to type 2 diabetes, comprising analyzing at
least one polynucleotide to detect at least one single nucleotide
polymorphism (SNP) selected from the group consisting of a T allele at
rs3760106, a G allele at rs2575390, a TT genotype of rs7404928, a A
allele of rs4787733 in a PKC-.beta.1 gene, and combinations thereof,
wherein the presence of said single nucleotide polymorphism indicates
that the subject is suffering from, at risk for, or suspected of
suffering from said diseases, disorder or condition.

2. The method of claim 1, wherein the method comprises detecting a
haplotype consisting of 3 variants or SNPs comprising variants at
rs3760106, rs7404928, and rs4787733; variants at rs2575390, rs7404928,
and rs4787733; or variants at rs3760106 and rs2575390, plus one at
rs7404928 or rs4787733.

3. The method of claim 1, wherein the method comprises detecting a
haplotype consisting of 4 variants or SNPs which are at rs3760106,
rs2575390, rs7404928 and rs4787733, respectively.

4. The method of claim 1 further comprising a step of obtaining a sample
from the subject.

5. The method of claim 1 wherein the sample is selected from the group
consisting of blood, semen, saliva, tears, urine, fecal material, sweat,
buccal cells, skin, hair and other nucleic acid-containing tissue.

6. The method of claim 1, wherein the subject is suffering from Type 2
Diabetes.

7. The method of claim 1, wherein said disease, disorder or condition is
ESRD.

8. A kit or an array for diagnosing a genetic predisposition in a subject
for a disease, disorder or condition a diabetic kidney complication
selected from the group consisting of end stage renal disease (ESRD) due
to type 2 diabetes, ESRD due to hypertension in type 2 diabetes, ESRD due
to type 1 diabetes; kidney disease of type 2 diabetes or type 1 diabetes;
cardiovascular disease due to type 2 diabetes or type 1 diabetes; and
cerebrovascular accident due to type 2 diabetes, comprising reagents for
detecting at least one single nucleotide polymorphism in a sample
containing at least one polynucleotide obtained from said subject,
wherein said single nucleotide polymorphism is selected from the group
consisting of T allele at rs3760106, G allele at rs2575390, TT genotype
of rs7404928, A allele of rs4787733 in PKC-.beta.1 gene, and combinations
thereof.

9. The kit or array of claim 8, wherein the kit or array comprises
reagents for detecting a haplotype consisting of 3 variants or SNPs
comprising variants at rs3760106, rs7404928, and rs4787733; variants at
rs2575390, rs7404928, and rs4787733; or variants at rs3760106 and
rs2575390, plus one at rs7404928 or rs4787733.

10. The kit or array of claim 8, wherein the kit or array comprises
reagents for detecting a haplotype consisting of 4 variants or SNPs which
are at rs3760106, rs2575390, rs7404928 and rs4787733, respectively.

11. The kit or array of claim 8 wherein the sample is selected from the
group consisting of blood, semen, saliva, tears, urine, fecal material,
sweat, buccal cells, skin, hair and other nucleic acid-containing tissue.

12. The kit or array of claim 8, wherein the subject is suffering from
Type 2 Diabetes.

13. A method for treating or preventing a disease, condition or disorder
in a subject having at least one single nucleotide polymorphism (SNP)
selected from the group consisting of T allele at rs3760106, G allele at
rs2575390, TT genotype of rs7404928, A allele of rs4787733 in PKC-.beta.1
gene, and combinations thereof, comprising administering to the subject a
compound counteracting the effect of any said polymorphism in the
subject, wherein said disease, condition or disorder comprises a diabetic
kidney complication selected from the group consisting of end stage renal
disease (ESRD) due to type 2 diabetes, ESRD due to hypertension in type 2
diabetes, ESRD due to type 1 diabetes; kidney diseases of type 2 diabetes
or type 1 diabetes; cardiovascular disease due to type 2 diabetes or type
1 diabetes; and cerebrovascular accident due to type 2 diabetes, and
wherein said compound comprises one or more agents for inhibiting at
least one said SNPs, and wherein the agent is selected from the group
consisting of inhibitory RNA, an antibody, an anti-sense nucleic acid,
and an agent or drug for reduction of blood pressure, glucose control,
lipid parameters, or modulating the renin-angiotensin system.

14. The method of claim 13, wherein said disease, disorder or condition
is ESRD.

15. The method of claim 1, wherein the cardiovascular disease due to type
2 diabetes or type 1 diabetes is selected from the group consisting of
atherosclerotic peripheral vascular disease, hypertension, ischemic
cardiomyopathy, and myocardial infarction due to type 2 diabetes or type
1 diabetes.

16. The kit or array of claim 8, wherein the cardiovascular disease due
to type 2 diabetes or type 1 diabetes is selected from the group
consisting of atherosclerotic peripheral vascular disease, hypertension,
ischemic cardiomyopathy, and myocardial infarction due to type 2 diabetes
or type 1 diabetes.

17. The method of claim 13, wherein the cardiovascular disease due to
type 2 diabetes or type 1 diabetes is selected from the group consisting
of atherosclerotic peripheral vascular disease, hypertension, ischemic
cardiomyopathy, and myocardial infarction due to type 2 diabetes or type
1 diabetes.

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to methods, kits and arrays for
diagnosing or detecting a subject's genetic susceptibility for a disease,
condition, or disorder in a subject, in particular for diabetic kidney
complications including end stage renal disease (ESRD), and other
cardiovascular diseases.

BACKGROUND OF THE INVENTION

[0002] Diabetic nephropathy is a leading cause of morbidity and mortality
in diabetic patients. With the rising epidemic of diabetes in both
developing and developed countries, diabetes is now the major
contributing cause of end stage renal disease (ESRD). Asia is in the
midst of an epidemic of type 2 diabetes (non-insulin dependent diabetes
mellitus)1 2 3. A recent large-scale epidemiology survey estimated
that there are 94.2 million adults affected by diabetes in China, with
another 148.2 million with prediabetes4. Renal failure or ESRD is an
important cause of mortality among patients with type 2 diabetes5 6.
Asian populations appear to be particularly at risk of diabetic kidney
disease (DKD). In the MAP study, microalbuminuria was present in 40% and
macroalbuminuria in 20% of Asian patients with type 2 diabetes and
hypertension7. Progression to ESRD can be effectively reduced by
aggressive reduction of multiple targets including blood pressure,
glucose control, lipid parameters as well as agents which modulate the
renin-angiotensin system. However, it is often not possible to predict
which subjects with diabetes are at increased risk of developing renal
complications, especially the ESRD.

[0003] Protein kinase C (PKC)-β is an important molecule involved in
cell signaling, and has been implicated in the development of diabetic
microvascular complications as well as diabetic cardiomyopathy14 15
16 17. Pharmacological inhibition of PKC-β was found to be effective
in normalizing haemodynamic changes, extracellular matrix (ECM)
accumulation, histological features of glomerular damage as well as
reduced albuminuria in diabetic animal models18 19 20. Mice that
lack PKC-β are protected from glomerular hypertrophy, oxidative
stress and albuminuria when exposed to hyperglycaemia21. In
randomized clinical trials, inhibitor of PKC-β decreased loss of
glomerular filtration rate and proteinuria in diabetic patients who are
optimally treated with ACEI or ARB22. These data support an
important role of PKC-β in the pathogenesis of DKD. Both isoforms of
PKC-β, PKC-βI and PKC-βII, are encoded by the PKC-β1
gene (PRKCB) on the short arm of chromosome 16. Type 1 diabetic patients
(insulin dependent diabetes mellitus) carrying 2 single nucleotide
polymorphism (SNP)s in the promoter region of the PRKCB had increased
risk of nephropathy23 24.

SUMMARY

[0004] The present inventors have surprisingly discovered that certain
single nucleotide polymorphisms (SNPs) within the PKC-β1 (PRKCB)
gene and related regulatory regions are associated with the development
of diabetic kidney complications comprising kidney diseases of type 2
diabetes or type 1 diabetes, end stage renal disease (ESRD) due to type 2
diabetes, ESRD due to hypertension in type 2 diabetes, ESRD due to type 1
diabetes; cardiovascular disease due to type 2 diabetes or type 1
diabetes such as atherosclerotic peripheral vascular disease,
hypertension, ischemic cardiomyopathy, and myocardial infarction due to
type 2 diabetes or type 1 diabetes; and cerebrovascular accident due to
type 2 diabetes. Therefore, the SNPs are useful for methods and kits of
diagnosing a genetic predisposition for the development of the diseases
in individuals, and further for the methods for treating or preventing
the diseases.

[0005] Accordingly, one aspect disclosed herein provides a method for
diagnosing a genetic predisposition in a subject for the diseases,
disorders or conditions selected from the group consisting of diabetic
kidney complications comprising kidney diseases of type 2 diabetes or
type 1 diabetes, end stage renal disease (ESRD) due to type 2 diabetes,
ESRD due to hypertension in type 2 diabetes, ESRD due to type 1 diabetes;
cardiovascular disease due to type 2 diabetes or type 1 diabetes such as
atherosclerotic peripheral vascular disease, hypertension, ischemic
cardiomyopathy, and myocardial infarction due to type 2 diabetes or type
1 diabetes; and cerebrovascular accident due to type 2 diabetes,
comprising analyzing at least one polynucleotide to detect a genetic
polymorphism selected from the group consisting of T allele at rs3760106,
G allele at rs2575390, TT genotype of rs7404928, A allele of rs4787733 in
PKC-β1 gene, and any combination thereof, wherein the present of at
least one said genetic polymorphism indicates the subject is suffering
from, at risk for, or suspected of suffering from said diseases,
disorders or conditions.

[0006] Another aspect disclosed herein provides a kit or array for
diagnosing a genetic predisposition in a subject for the diseases,
disorders or conditions selected from the group consisting of diabetic
kidney complications comprising kidney diseases of type 2 diabetes or
type 1 diabetes, especially end stage renal disease (ESRD) due to type 2
diabetes, ESRD due to hypertension in type 2 diabetes, ESRD due to type 1
diabetes; cardiovascular disease due to type 2 diabetes or type 1
diabetes such as atherosclerotic peripheral vascular disease,
hypertension, ischemic cardiomyopathy, and myocardial infarction due to
type 2 diabetes or type 1 diabetes; and cerebrovascular accident due to
type 2 diabetes, and the kit or array comprises reagents for detecting a
genetic polymorphism in a sample containing at least one polynucleotide
obtained from the subject.

[0007] Yet another aspect disclosed herein provides a method for treating
or preventing a disease, condition or disorder selected from the group
consisting of diabetic kidney complications comprising kidney diseases of
type 2 diabetes or type 1 diabetes, especially end stage renal disease
(ESRD) due to type 2 diabetes, ESRD due to hypertension in type 2
diabetes, ESRD due to type 1 diabetes; cardiovascular disease due to type
2 diabetes or type 1 diabetes such as atherosclerotic peripheral vascular
disease, hypertension, ischemic cardiomyopathy, and myocardial infarction
due to type 2 diabetes or type 1 diabetes; and cerebrovascular accident
due to type 2 diabetes in a subject having at least one of the genetic
polymorphisms as detected in the present invention, comprising
administering to the subject a compound counteracting the effect of any
said polymorphism in the subject, wherein said disease, condition or
disorder is selected from the group consisting of diabetic kidney
complications comprising kidney diseases of type 2 diabetes or type 1
diabetes, especially end stage renal disease (ESRD) due to type 2
diabetes, ESRD due to hypertension in type 2 diabetes, ESRD due to type 1
diabetes; cardiovascular disease due to type 2 diabetes or type 1
diabetes such as atherosclerotic peripheral vascular disease,
hypertension, ischemic cardiomyopathy, and myocardial infarction due to
type 2 diabetes or type 1 diabetes; and cerebrovascular accident due to
type 2 diabetes.

[0008] Further scope of the applicability of the present invention will
become apparent from the detailed description and drawings provided
below. It should be understood, however, that the following detailed
description and examples, while indicating embodiments of the invention,
are given by way of illustration only, since various changes and
modifications within the spirit and scope of the invention will become
apparent to those skilled in the art from the following detailed
description.

[0009] All publications, patents, patent applications and other references
cited in this application are herein incorporated by reference in their
entirety as if each individual publication, patent, patent application or
other reference were specifically and individually indicated to be
incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1. Cumulative probability of new-onset ESRD according to
number of risk alleles, adjusted for mean values of the conventional risk
factors (sex, age and duration of diabetes, systolic and diastolic blood
pressure, HbA1c, total cholesterol, natural logarithm of triglycerides,
eGFR, natural logarithm of AER, retinopathy (present/absent), the use of
drugs (yes/no)). Three (rs3760106 with dominant model, rs7404928 with
recessive model and rs4787733 with additive model) significant and
independent (r2<0.8) SNPs were selected to calculate the number
of risk alleles for ESRD. The total number of carriers for the risk
allele categories are indicated within the parentheses. The number of
subjects exposed to risk at each follow-up period time point stratified
by genotypes are indicated below the horizontal axis.

DEFINITIONS

[0011] As used herein the terms "patient" and "subject" are not limited to
human beings, but are intended to include all vertebrate animals in
addition to human beings. In some embodiments disclosed herein, the terms
"patient" and "subject" refer to any people or any ethnic groups in the
word, such as Asian ancestry, including Chinese ancestry or descent. In
other embodiments, the patient is a patient of type 2 diabetes, such as a
patient of type 2 diabetes of Chinese ancestry.

[0012] As used herein the terms "genetic predisposition", "genetic
susceptibility" and "susceptibility" all refer to the likelihood that an
individual subject is suffering from, at risk for, or suspected of
suffering from a particular disease, condition or disorder. For example,
a subject with an increased susceptibility or predisposition will be more
likely than average to develop a disease, while a subject with a
decreased predisposition will be less likely than average to develop the
disease.

[0013] As used herein, "gene" means any amount of nucleic acid material
that is sufficient to encode a transcript or protein having the function
desired. Thus, it includes, but is not limited to, genomic DNA, cDNA,
RNA, and nucleic acid that are otherwise genetically engineered to
achieve a desired level of expression under desired conditions.
Accordingly, it includes fusion genes (encoding fusion proteins), intact
genomic genes, and DNA sequences fused to heterologous promoters,
operators, enhancers, and/or other transcription regulating sequences.
The term refers to an entirety containing entire transcribed region and
all regulatory regions of a gene. The transcribed region of a gene
including all exon and intron sequences of a gene including alternatively
spliced exons and introns so the transcribed region of a gene contains in
addition to polypeptide encoding region of a gene also regulatory and 5'
and 3' untranslated regions present in transcribed RNA.

[0014] As used herein, the terms "single nucleotide polymorphism", "SNP"
and "genetic polymorphism" is a DNA sequence variation or a genetic
variant that occurs when a nucleotide, e.g., adenine (A), thymine (T),
cytosine (C), or guanine (G), in the genome sequence is altered to
another nucleotide. SNPs are occasional variations in DNA sequence; the
vast majority of the DNA sequence is identical among all humans, such as
Accession Number RefSeq NM--002738 for the protein kinase C beta 1
gene of the invention. SNPs or other variants may also be found in
genomic regions that do not contain genes. They represent a genomic hot
spot responsible for the genetic variability among humans. The term SNP
can be inter used with the term "genetic variant" or "variant" which is
present at a particular genetic locus in at least one individual in a
population and that differs from the corresponding reference type in the
vast majority of the DNA sequence.

[0015] As used herein, the term "haplotype" refers to any combination of
genetic variants or markers ("alleles") usually inherited together. A
haplotype can comprise two or more alleles and the length of a genome
region comprising a haplotype may vary from few hundred bases up to
hundreds of kilobases. As it is recognized by those skilled in the art,
the same haplotype can be described differently by determining the
haplotype defining alleles from different nucleic acid strands. For
example, the haplotype TTA defined by the SNP markers of this invention
is the same as haplotype TAA in which the alleles are determined from the
other strand. The haplotypes described herein are differentially present
in the patients with increased risk of developing one or more of the
aforementioned disease or complications. Therefore, these haplotypes have
diagnostic value for risk assessment, diagnosis and prognosis of kidney
disease and complications thereof. Detection of haplotypes can be
accomplished by methods known in the art used for detecting nucleotides
at polymorphic sites.

[0016] As used in this application, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates otherwise.
In particular, where the indefinite article is used, the specification is
to be understood as contemplating plurality as well as singularity,
unless the context requires otherwise.

[0017] As used herein, the term "about" or "approximately" when used in
conjunction with a number refers to any number within 1, 5 or 10% of the
referenced number.

[0018] Reference throughout this specification to "one embodiment", or "an
embodiment", or "in another embodiment", or "some embodiments", or "in
certain embodiments" means that a particular referent feature, structure,
or characteristic described in connection with the embodiment is included
in at least one embodiment. Thus, the appearance of the phrases "in one
embodiment", or "in an embodiment", or "in another embodiment", or "in
some embodiments" in various places throughout this specification are not
necessarily all referring to the same embodiment. Furthermore, the
particular features, structures, or characteristics may be combined in
any suitable manner in one or more embodiments.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] One aspect disclosed herein provides a method for diagnosing a
genetic predisposition in a subject for the diseases, disorders or
conditions selected from the group consisting of diabetic kidney
complications comprising kidney diseases of type 2 diabetes or type 1
diabetes, end stage renal disease (ESRD) due to type 2 diabetes, ESRD due
to hypertension in type 2 diabetes, ESRD due to type 1 diabetes;
cardiovascular disease due to type 2 diabetes or type 1 diabetes such as
atherosclerotic peripheral vascular disease, hypertension, ischemic
cardiomyopathy, and myocardial infarction due to type 2 diabetes or type
1 diabetes; and cerebrovascular accident due to type 2 diabetes. The
method for diagnosing a genetic predisposition in a subject for the
diseases, disorders or conditions comprises analyzing at least one
polynucleotide to detect a genetic polymorphism selected from the group
consisting of T allele at rs3760106, G allele at rs2575390, TT genotype
of rs7404928, A allele of rs4787733 in PKC-β1 gene, and any
combination thereof, wherein the present of at least one said genetic
polymorphism indicates the subject is suffering from, at risk for, or
suspected of suffering from said diseases, disorders or conditions.

[0020] In an embodiment, the method further comprises a step for obtaining
the sample containing at least one polynucleotide from the subject. In
other embodiment, the method can be performed in vitro where the sample
has been collected and is outside of the subject.

[0021] In some embodiments, the method comprises analyzing at least one
polynucleotide to detect at least one genetic polymorphism selected from
the group consisting of T allele at rs3760106, G allele at rs2575390, TT
genotype of rs7404928, and A allele of rs4787733 in PKC-β1 gene.

[0022] In one embodiment, the method comprises detecting a haplotype
consisting of 3 variants or SNPs, such as variants at rs3760106,
rs7404928, and rs4787733 (i.e. TTA); variants at rs2575390, rs7404928,
and rs4787733 (i.e. GTA). In another preferred embodiments, the method of
the invention comprises detecting a haplotype consisting of 3 variants or
SNPs consisting of those at rs3760106 and rs2575390, and an additional
one which is at rs7404928 or rs4787733.

[0023] In yet another embodiment disclosed herein, the method comprises
detecting a haplotype consisting of 4 variants or SNPs which are at
rs3760106, rs2575390, rs7404928 and rs4787733, respectively.

[0024] Another aspect disclosed herein provides a kit or array for
diagnosing a genetic predisposition in a subject for the diseases,
disorders or conditions selected from the group consisting of diabetic
kidney complications comprising kidney diseases of type 2 diabetes or
type 1 diabetes, especially end stage renal disease (ESRD) due to type 2
diabetes, ESRD due to hypertension in type 2 diabetes, ESRD due to type 1
diabetes; cardiovascular disease due to type 2 diabetes or type 1
diabetes such as atherosclerotic peripheral vascular disease,
hypertension, ischemic cardiomyopathy, and myocardial infarction due to
type 2 diabetes or type 1 diabetes; and cerebrovascular accident due to
type 2 diabetes, comprising reagents for detecting a genetic polymorphism
in a sample containing at least one polynucleotide obtained from said
subject.

[0025] Yet another aspect disclosed herein provides a method for treating
or preventing a disease, condition or disorder selected from the group
consisting of diabetic kidney complications comprising kidney diseases of
type 2 diabetes or type 1 diabetes, especially end stage renal disease
(ESRD) due to type 2 diabetes, ESRD due to hypertension in type 2
diabetes, ESRD due to type 1 diabetes; cardiovascular disease due to type
2 diabetes or type 1 diabetes such as atherosclerotic peripheral vascular
disease, hypertension, ischemic cardiomyopathy, and myocardial infarction
due to type 2 diabetes or type 1 diabetes; and cerebrovascular accident
due to type 2 diabetes in a subject having at least one of the genetic
polymorphisms as detected in the present invention. The method for
treating or preventing the disease, condition or disorder as disclosed
herein comprises administering to the subject a compound counteracting
the effect of any said polymorphism in the subject, wherein the disease,
condition or disorder is selected from the group consisting of diabetic
kidney complications comprising kidney diseases of type 2 diabetes or
type 1 diabetes, especially end stage renal disease (ESRD) due to type 2
diabetes, ESRD due to hypertension in type 2 diabetes, ESRD due to type 1
diabetes; cardiovascular disease due to type 2 diabetes or type 1
diabetes such as atherosclerotic peripheral vascular disease,
hypertension, ischemic cardiomyopathy, and myocardial infarction due to
type 2 diabetes or type 1 diabetes; and cerebrovascular accident due to
type 2 diabetes.

[0026] In an embodiment of the method for the treatment of the invention,
the compound comprises agents for inhibit at least one said SNPs selected
from inhibitory RNA, antibody, anti-sense nucleic acids, as well as
agents or drugs for reduction of blood pressure, glucose control, lipid
parameters, and those for modulating the renin-angiotensin system. It
would be appreciated by a person skilled in the art that any agents which
can treat, alleviate the symptoms of the diseases mentioned above, or
inhibit the progress of DKD or ESRD can be used for administration to the
subject.

[0027] Although the numerical chromosomal position of a SNP may still
change upon annotating the current human genome build the SNP
identification information such as variable alleles and flanking
nucleotide sequences assigned to a SNP will remain the same. Those
skilled in the art will readily recognize that the analysis of the
nucleotides present in one or more SNPs set forth herein in an
individual's nucleic acid can be done by any method or technique capable
of determining nucleotides present in a polymorphic site using the
sequence information assigned in prior art to the rs IDs of the SNPs
listed disclosed herein. As it is obvious in the art the nucleotides
present in polymorphisms can be determined from either nucleic acid
strand or from both strands.

[0028] Diagnostic kits (e.g. reagent kits) or arrays disclosed herein
comprise reagents or materials, and optionally protocols or instructions
for assessing one or more SNPs to make risk assessment, diagnosis or
prognosis of a diabetes related complication and optimized therapeutic
suggestions. Useful reagents and materials for kits include, but are not
limited to PCR primers, hybridization probes and primers as described
herein (e.g., labeled probes or primers), allele-specific
oligonucleotides, reagents for genotyping SNP markers, reagents for
detection of labeled molecules, restriction enzymes (e.g., for RFLP
analysis), DNA polymerases, RNA polymerases, DNA ligases, marker enzymes,
antibodies which bind to altered or to non-altered (native) a
polypeptide, means for amplification of nucleic acids fragments from one
or more SNPs selected from, means for analyzing the nucleic acid sequence
of one or more diabetes-complication or ESRD related SNPs, or means for
analyzing the sequence of one or more amino acid residues of polypeptides
encoded by genes comprising such SNPs, etc. In one embodiment, a kit for
diagnosing susceptibility to a diabetes-related complication or ESRD
comprises primers and reagents for detecting the nucleotides present in
one or more SNP markers selected from rs3760106, rs2575390, rs7404928 and
rs4787733 in PKC-β1 gene in individual's nucleic acid.

Complications Associated with Diabetes

[0029] More than 90% of people diagnosed with diabetes, especially type 2
diabetes, carry a number of potential complications. Half of the people
affected by diabetes die from complications resulting from the disease.

[0030] The complications involved, diagnosed or detected by the methods,
kits or arrays of the present invention include, but are not limited to:

1. Cardiovascular Disease

[0031] Cardiovascular disease is the overwhelming cause of
diabetes-related deaths. With the risk for stroke or myocardial
infarction elevated by more 2 times in persons with diabetes, a majority
of deaths among people with diabetes occurs from heart disease or stroke,
considered as major macrovascular complications. Diabetic patients or
subjects (especially type 2 diabetes) with reduced estimated glomerular
filtration rate (eGFR) will be at high risk of developing cardiovascular
diseases, even heart failure.

2. Diabetic Nephropathy

[0032] End-stage renal disease (ESRD) occurs when the kidneys cease to
function, which ultimately leads to the need for a transplant or regular
dialysis, both extremely costly procedures. Diabetes is responsible for
about 50% of the cases of ESRD as a consequence of microvascular damage
of the kidney.

3. Diabetic Retinopathy

[0033] Diabetes is also the leading cause of blindness in people. Diabetic
retinopathy is considered as one type of microvascular complication.

4. Diabetic Neuropathy

[0034] It is estimated that more than 50% of people with diabetes may also
suffer from nervous system damage, causing impaired sensation or pain in
the feet or hands, slowed digestion of food in the stomach, carpal tunnel
syndrome, and other nerve problems. In the severe cases of diabetic
neuropathy, usually combined with peripheral vessel macro and
microvascular disease, patients may have to undergo lower-extremity
amputations.

[0036] In a preferred embodiment of the invention, the complications
involved are selected from the group consisting of diabetic kidney
complications such as ESRD, and cardiovascular diseases.

Preparation of Samples

[0037] The presence of genetic variants or SNPs in the PKC-β1 gene
that may affect susceptibility to disease is determined by screening at
least one nucleic acid sequence from an individual subject for such
variants.

[0038] The nucleic acid sequence can be DNA or RNA. For the assay of
genomic DNA, virtually any biological sample containing genomic DNA (e.g.
not pure red blood cells) can be used. For example, and without
limitation, genomic DNA can be conveniently obtained from blood, semen,
saliva, tears, urine, fecal material, sweat, buccal cells, skin or hair.
For assays using cDNA or mRNA, the target nucleic acid must be obtained
from cells or tissues that express the target sequence. One preferred
source and quantity of DNA is 3 to 30 ml of anticoagulated whole blood,
since enough DNA can be extracted from leukocytes in such a sample to
perform many repetitions of the analysis contemplated herein.

[0039] Many of the methods described herein require the amplification of
DNA from target samples. This can be accomplished by any method known in
the art but preferably is by the polymerase chain reaction (PCR).
Optimization of conditions for conducting PCR must be determined for each
reaction and can be accomplished without undue experimentation by one of
ordinary skill in the art. In general, methods for conducting PCR can be
found in Ausbel et al., eds., Short Protocols in Molecular Biology,
3rd ed., Wiley, 1995; and Innis et al., eds., PCR Protocols,
Academic Press, 1990.

[0041] The polymorphism detection involves determining which form of the
polymorphism is present in individuals for diagnostic or epidemiological
purposes. The detection technology comprises PCR-based technology, or
methods involving primer extension of multiplex products with detection
by MALDI-TOF mass spectroscopy on a MassARRAY platform (Sequenom, San
Diego, Calif.). Several methods have been developed to detect known SNPs.
Many of these assays have been reviewed by Landegren et al., Genome Res.,
8:769-776, 1998, and will only be briefly reviewed here.

[0045] The joining by DNA ligases of two oligonucleotides hybridized to a
target DNA sequence is quite sensitive to mismatches close to the
ligation site, especially at the 3' end. This sensitivity has been
utilized in the oligonucleotide ligation assay (OLA; Landegren et al.,
Science, 241:1077-1080, 1988) and the ligase chain reaction (LCR; Barany,
Proc. Natl. Acad. Sci. USA, 88:189-193, 1991).

[0046] In another method for the detection of SNPs termed minisequencing,
the target-dependent addition by a polymerase of a specific nucleotide
immediately downstream (3') to a single primer is used to determine which
allele is present (U.S. Pat. No. 5,846,710).

[0047] The above discussion of methods for the detection of SNPs is
exemplary only and is not intended to be exhaustive. Those of ordinary
skill in the art will be able to envision other methods for detection of
SNPs that are within the scope and spirit of the present invention.

SNPs

[0048] In the present invention, PRKCB gene (accession number:
NM--002738) is selected to determine the susceptibility to a
disease, condition, or disorder in a subject, especially the diseases,
disorders or conditions as described herein. In an embodiment, the
tagging SNPs comprising at least one of rs3760106, rs2575390, rs7404928
and rs4787733, are responsible for the susceptibility to the diseases,
disorders or conditions in the subject. The methods, kits and arrays as
described herein relate to detection of SNPs at genetic loci of
rs3760106, rs2575390, rs7404928, and rs4787733. In one embodiment, the
methods, kits and arrays as described herein relate to detection of SNPs
at the genetic loci comprising at least one, at least two or at least
three of: rs3760106, rs2575390, rs4787733, and rs7404928.

[0049] The SNP is associated with a genetic predisposition for a disease,
condition or disorder comprising but not limited to non-insulin dependent
diabetes, insulin dependent diabetes, end stage renal disease due to
non-insulin dependent diabetes, hypertension due to non-insulin dependent
diabetes, end stage renal disease due to hypertension of non-insulin
dependent diabetes, atherosclerotic peripheral vascular disease due to
non-insulin dependent diabetes, and end stage renal disease due to
insulin dependent diabetes.

[0050] Although the methods or kits disclosed herein can be performed in
sample obtained from a subject with or without diabetes, it is preferred
to perform them in a subject who is suffering from Type 2 diabetes. The
subject can be of any ethnic group, such as Asian, including Chinese
descent.

[0051] After adjustment for age and sex, we surprised find that the risk
for having the genetic susceptibility increases progressively and
significantly with increasing number of risk genotypes or SNPs. Patients
with 3 or 4 risk genotypes or SNPs had 1.5-2.0 fold increased risk for
DKD or ESRD, compared to those patients with 0 or 1 risk genotype. In
particular, the use of a haplotype consisting of the 3 variants (such as
rs3760106, rs7404928, and rs4787733) indicates that individuals carrying
the risk-conferring haplotype (such as TTA for rs3760106, rs7404928, and
rs4787733) is associated with about 1.5 to 2.0 fold or more (for TTA for
rs3760106, rs7404928, and rs4787733, about 1.89 fold) increased risk of
end stage renal disease compared with those patients with 0 or 1 risk
genotype, whilst carriers of the protective (such as CCG for rs3760106,
rs7404928, and rs4787733) alleles at the 3 respective variants are
protected against the development of diabetic kidney complications.

[0052] The risk of developing end stage renal disease increases with
increasing number of risk alleles within the PRKCB gene. For example, the
adjusted risk for end stage renal disease is more than 6 (95% CI,
2.00-18.31) for patients with 4 risk alleles (i.e. rs3760106, rs2575390,
rs4787733, and rs7404928) compared with patients with 0 or 1 risk allele.
Incidence of end stage renal disease among individuals carrying 4 risk
alleles is increased compared with those carrying 0 or 1 risk allele. For
example, incidence of end stage renal disease was 4.4 per 1000 person
years (95% CI, 0.5-8.2) among individuals with 0 or 1 risk allele
compared with 20.0 per 1000 person-years (95% CI, 8.8-31.1) in those
carrying the 4 risk alleles (i.e. rs3760106, rs2575390, rs4787733, and
rs7404928).

[0053] It is understood that one or more genotypes mentioned above can be
used to develop arrays that are used in conjunction with other known
clinical, biochemical and genetic for predicting the risk of diabetic
complications including nephropathy and ESRD in diabetic patients of
Chinese ancestry or other ethnic groups in the world, and these genotypes
or equivalent arrays thereof can be used to identify at risk subjects for
diabetes and/or diabetic ESRD for risk modification using a multifaceted
approach including intensive monitoring, pharmacological and
non-pharmacological therapy.

[0054] The present invention will be further described with the following
Examples.

Example

Methods

Study Participants

[0055] Our cohort consists of 1338 unrelated patients with type 2 diabetes
from the Hong Kong Diabetes Registry (HKDR) enrolled between 1995 and
1998. All subjects were of southern Han Chinese ancestry residing in Hong
Kong. Patients with classical type 1 diabetes (defined as acute
presentation with diabetic ketoacidosis, heavy ketonuria (>3+), or
continuous requirement of insulin within 1 year of diagnosis) were
excluded. In addition, patients with incomplete clinical information
(N=4), follow-up period less than 3 years (N=145), requirement of
dialysis at baseline (N=1), or had ESRD defined as estimated glomerular
filtration rate (eGFR) less than 15 ml/min per 1.73 m2 at enrollment
(N=16) were excluded. The study design, ascertainment, inclusion criteria
and phenotyping of the study subjects have been described6 27. For
this analysis, a total of 1172 unrelated patients with type 2 diabetes
were included (mean (±SD) age 56.0±11.9 years, 41.3% male, disease
duration 8.5±6.7 years).

[0056] The validation cohort consisted of additional subjects recruited
from the Hong Kong Diabetes Registry. Subjects included in the
replication cohort were recruited into the Registry in an identical
manner to the original cohort, but were recruited from 1998 onwards.
Patients with chronic kidney disease at enrollment were excluded in the
validation cohort. For the cross-sectional study on association between
PRKCB genotype and quantitative measures of renal function, we recruited
1,892 unrelated Chinese type 2 diabetic patients from the inpatient
database of Shanghai Diabetes Institute28. This study was approved
by the Clinical Research Ethics Committee of the Chinese University of
Hong Kong and the Ethics Committee of the Shanghai Jiaotong University.
Written informed consent was obtained from all participants.

Clinical Studies and Outcomes

[0057] All study participants were examined in the morning after an
overnight fast. Anthropometric parameters including body weight and
height, waist circumference and blood pressure were measured. Fasting
blood samples were collected for measurement of plasma glucose,
HbA1c, and lipid profile (total cholesterol (TC), triglycerides
(TG), HDL and LDL cholesterol). Hypertension was defined as blood
pressure≧130/85 mmHg or use of anti-hypertensive medications (not
including angiotensin-coverting enzyme inhibitors (ACEIs) and angiotensin
receptor blockers (ARBs)), or use of ACEIs or ARBs. A timed urine
collection (4- or 24-hours) was used for measurement of urinary albumin
excretion rate (AER). Glomerular filtration rate (eGFR) was estimated
using the abbreviated formula developed by the Modification of Diet in
Renal Disease (MDRD) further adjusted for the Chinese ethnicity:
eGFR=186×[SCR×0.011]-1.154×[age]-0.203.t-
imes.[0.742 if female]×[1.233 if Chinese] where ScR is serum
creatinine expressed as μmol/l and 1.233 is the adjusting coefficient
for Chinese population29. Use of medications, including oral blood
glucose lowering agents and insulin treatment were also recorded for all
study subjects. Anti-hypertensive medications included all classes of
drugs that are indicated for hypertension, other than ACEI/ARB.
Lipid-lowering drugs included statins and fibrates.

[0058] All clinical endpoints including hospital admissions and mortality
were censored on 30 Jul., 2005 according to the databases from the
Hospital Authority Central Computer System, which records admissions to
all public hospitals. These databases, including the Hong Kong Death
Registry, were matched by a unique identification number, the Hong Kong
Identity Card number, which is compulsory for all residents in Hong Kong
and used by all government departments and major organizations. Using the
International Classification of Diseases--9th Revision code, ESRD
was defined as 1) death due to diabetes with renal manifestations (Code
250.4), chronic renal failure (Code 585) or unspecified renal failure
(Code 586) or 2) nonfatal chronic renal failure (Code 585) or unspecified
renal failure (Code 586) or 3) dialysis (ICD-9 procedure code: 39.95) or
peritoneal dialysis (ICD-9 procedure code: 54.98) or 4) follow-up
eGFR<15 ml/min per 1.73 m2.

[0060] Based on the Phase III HapMap database (HapMap release 27 [February
2009], NCBI Build 36; dbSNP b126) specific for the Han Chinese (CHB)
population, we selected 18 SNPs from a 388 kb region (chromosome
16:23,752,823-24,141,063 bp) spanning 2 kb upstream and downstream of
PRKCB (accession number: NM--002738). Among these SNPs, 12 tagging
SNPs (rs7404928, rs3785394, rs3785391, rs120908, rs4788423, rs4787733,
rs198198, rs380932, rs429342, rs1015408, rs3785380, rs3785378) with
r2<0.8 and minor allele frequency (MAF)≧0.05 were selected
using the Tagger algorithm in the Haploview (v 4.1) program. Under the
"common disease-common variant hypothesis", common diseases are
hypothesized to have common causal variants (those with minor allele
frequency (MAF)≧0.05), so we focused on investigating the effects
of common genetic variants in this study. Also, SNPs with r2>0.8
are considered redundant as 80% of their information overlapped, so
selecting just one of them would be more cost-effective. In addition, we
selected six SNPs (rs3760106, rs2575390, rs3900007, rs3900008, rs432998
and rs3729904) with suggestive evidence of association from previous
studies23 24, so a total of 18 SNPs were genotyped in all study
subjects in 2009 using genomic DNA.

[0061] Genotyping was performed at the McGill University and Genome Quebec
Innovation Centre using primer extension of multiplex products with
detection by MALDI-TOF mass spectroscopy on a Sequenom MassARRAY platform
(San Diego, Calif., USA). All SNPs were in Hardy-Weinberg equilibrium
(P>0.05), as assessed by the exact test of PLINK31. The overall
genotype call rate was 99.3%. Genotyping accuracy was demonstrated by
showing>99.6% overall concordance rate in 14 blinded duplicate
samples. Samples from Shanghai were genotyped using MassARRAY iPLEX
system (MassARRAY Compact Analyzer, Sequenom, San Diego, Calif., USA).

Bioinformatic Analyses and Functional Annotation

[0062] To annotate the possible underlying functions of the genotyped loci
within PRKCB, the data of chromatin structure, evolutionary conserved
sequence, CpG islands, and cis-regulatory elements were assessed using
the following available bioinformatics tools. The genomic locations
(Ensembl Build 40, NBCI v36, hg18) of those SNPs were mapped from
dbSNP130 and tracked on the University of California Santa Cruz human
genome browser. The data of CpG islands, UW histone modification
region32 and PRKCB coding region were selected on the UCSC genome
browser. The conserved sequence in the promoter region was identified by
cisRED database33. The transcriptional regulatory modules within the
gene were predicted by PReMOD34.

Statistical Analysis

[0063] All data are expressed as percentage, mean±SD or median
(interquartile range), as appropriate. Triglycerides and AER were natural
log-transformed due to skewed distributions. Between-group comparisons
were performed by chi-squared test for categorical variables, and
unpaired Student's t-test or the Wilcoxon Rank Sum test for continuous
variables.

[0064] The relationships between PRKCB polymorphisms under additive,
dominant and recessive genetic models and ESRD were tested by Cox
proportional hazard regression model with adjustment for conventional
risk factors at baseline including sex, age, duration of diabetes,
systolic and diastolic blood pressure, HbA1c, total cholesterol,
natural logarithms of triglycerides and AER, eGFR, retinopathy
(present/absent) and use of drugs (yes/no). Additive model assumes that
the casual allele exerts an additive effect, so that carriers with 0, 1,
or 2 risk alleles would have no, some and the most causal effect
respectively. Dominant model assumes that all carriers with the casual
allele (both homozygote and heterozygote) would have a causal effect,
while recessive model assumes that only carriers with two causal alleles
(homozygote) would have a causal effect. Hazard ratios (HRs) with 95%
confidence intervals (CIs) were presented. We corrected for multiple
comparisons of SNPs under additive, dominant and recessive genetic models
using the permutation method rather than the false discovery rate
approach. The largest test statistic obtained from the three models was
chosen (MAX statistic)35. Since the distribution of the MAX
statistic under the null hypothesis is unknown, experiment-wise
significance was estimated from the empirical distribution of the MAX
statistic after performing 10,000 permutations of genotypes for all 18
SNPs.

[0065] Pairwise linkage disequilibrium measures were computed in all
samples using Haploview. Haplotype frequencies between patients with and
without new-onset end stage renal disease were compared using
haplotype-specific test implemented in Haploview. Hazard ratios (HRs)
with 95% confidence intervals (CIs) were calculated for risk association
of ESRD with various haplotypes. To assess interaction effects between
pairwise SNPs on outcome variables, Cox proportional hazard regression
model including the main and interaction effects of SNPs with covariates
was applied.

[0066] The joint effect of the three PRKCB polymorphisms (rs3760106 with
dominant model, rs7404928 with recessive model and rs4787733 with
additive model) for ESRD risks were shown by Kaplan-Meier curves adjusted
for conventional risk factors at baseline, for which the cumulative
probability of new-onset events according to number of risk alleles was
estimated. The significance of the trend was tested by Cox regression
using the categories of risk allele carried as an independent variable.

[0067] All statistical analyses were performed using SAS v.9.1 (SAS
Institute, Cary, N.C., USA) or SPSS for Windows v.15 (SPSS, Chicago,
Ill., USA) unless specified otherwise. A two-tailed P value<0.05 was
considered statistically significant. We estimated the posterior study
power using genetic power calculator36. Assuming dominant models
with minor allele frequencies of 0.07, our sample size have 95% power to
detect a minimal HR 2.25 for ESRD, at a level of 0.05.

Results

Identification of Genetic Variants

[0068] We genotyped 18 SNPs spanning across the PRKCB gene in 1172 type 2
diabetic patients, including four SNPs in the promoter region, two SNPs
in the coding exons and six SNPs in the non-coding intron region (Table
2). The gene structure and the location of SNPs are shown in eFigure 1.
All SNPs were in modest linkage disequilibrium with r2<0.8 in our
population except for four pairs of SNPs (rs3760106 and rs2575390;
rs3900007 and rs3900008; rs3785394 and rs3785391; rs3785380 and
rs3785378). The latter two pairs of SNPs demonstrated stronger LD in our
population as compared to the HapMap CHB population (for rs3785394 and
rs3785391, r2=0.81 in present study and 0.78 in HapMap CHB data; for
rs3785380 and rs3785378, r2=0.94 in present study and 0.78 and
HapMap CHB data).

Clinical Characteristics of Study Participants

[0069] During the study period, 90 patients developed ESRD. The mean
follow-up period for ESRD was 7.9±1.9 years. Patients with new-onset
ESRD were older and had higher HbA1C, TC, TG levels, LDL-C, systolic
and diastolic blood pressure, AER, rates of hypertension and retinopathy
and were more likely to be treated with insulin, ACEI and lipid-lowering
drugs at baseline than those without. In addition, patients with
new-onset ESRD had lower eGFR (Table 1).

Association Between Variants of PRKCB and ESRD

[0070] The frequencies of the minor T-allele of rs3760106 and G-allele of
rs2575390 (r2=0.99) were markedly different between patients with
(12.2%) and without incident ESRD (7%).

[0071] In the Cox-regression model after adjustment for conventional risk
factors, four SNPs were significantly associated with ESRD (P<0.05)
(Table 2). The T-allele at rs3760106 and the G-allele at the closely
linked SNP rs2575390 (r2=0.98) were strongly associated with
increased risk for ESRD (HR (95% C.I.)=2.25 (1.31-3.87), P=0.0034 for
rs3760106 in dominant model; HR (95% C.I.)=2.26 (1.31-3.88), P=0.0033 for
rs2575390 in dominant model). We also observed nominal associations for
ESRD with the common TT genotype of rs7404928 (HR (95% C.I.)=1.59
(1.01-2.52), P=0.0471 in recessive model) and the major A-allele of
rs4787733 (HR (95% C.I.)=1.78 (1.03-3.09), P=0.0399 in additive model).
The 2 SNPs rs3760106 (P=0.0299) and rs2575390 (P=0.0298) remained
significant after correction for multiple comparisons.

[0073] We further examined possible two-way epistasis of PRKCB
polymorphisms on ESRD, but failed to observe any evidence of interaction
(data not shown). However, there were significantly increased risks for
ESRD with increasing number of risk alleles (P=0.0007 for ESRD) in the
joint effect analysis of three (rs3760106 with dominant model, rs7404928
with recessive model and rs4787733 with additive model) significant and
independent (r2<0.8) SNPs (FIG. 1 and table 4). The adjusted risk
for ESRD was 6.044 (95% C.I. 2.00-18.31) in patients with four risk
alleles compared to patients with one or no risk allele. Apart from
presence of risk variants of PRKCB, other independent risk factors
identified for development of ESRD were elevated HbA1c, decrease in eGFR,
elevated log-transformed AER and presence of retinopathy (table 4). Among
our cohort of subjects who were free of ESRD at baseline, new ESRD events
occurred in 90 (7.7%) subjects during a maximum of 9-year follow-up,
giving an annualized incidence of 9.7/1,000 person-years (95%
CI=7.7-11.7). The incidence was 4.4/1,000 person-years (95% CI=0.5-8.2)
for subjects carrying 0 or 1 risk alleles (12.3% of the cohort), compared
to 20.0/1,000 (95% CI=8.8-31.1) in those carrying 4 risk alleles (6.9% of
the cohort).

Validation of Variants in PRKCB and Development of Diabetic Kidney Disease

[0074] In order to validate the association between PRKCB polymorphisms
and diabetic kidney disease, we examined the effects of variants in PRKCB
in additional subjects from the Hong Kong Diabetes Registry. All subjects
in the validation cohort were recruited subsequent to the initial
discovery cohort. The baseline characteristics of patients in the
validation cohort were summarized in eTable 1. Patients in the validation
cohort were older, with significantly shorter duration of diabetes and
follow-up compared to the original cohort, and with significantly more
patients treated with ACE inhibitors. As a result, a smaller proportion
of the patients in this cohort developed ESRD during the follow-up period
(253 subjects out of total 3677 subjects, 6.9%) compared with the
original cohort. In order to examine the effects of variants of PRKCB on
development of DKD in this cohort, we selected patients from this cohort
with early-onset disease (defined as age of onset <45 years) who were
free of chronic kidney disease at baseline and identified patients who
progressed to chronic kidney disease during the follow-up period. The
characteristics of this cohort of patients genotyped were summarized in
eTable 1. Out of a total of 1049 patients, 151 (14.3%) developed CKD
during the follow-up period. Both the T allele of the rs3760106 variant
and the G allele of the rs2575390 variant were found to be significantly
associated with development of CKD in this cohort, with HR of 1.68 (95%
CI 1.1-2.57) and 1.62 (95% CI 1.07-2.47) respectively (eTable2). Although
rs7404928 and the rs4787733 variants did not reach statistical
significance for association with chronic kidney disease, the at-risk
variants increased risk of developing renal dysfunction in the same
direction using the same genetic model as in the original discovery
cohort.

Relationship Between Variants of PRKCB and eGFR

[0075] In order to better understand the relationship between genetic
variants of PRKCB and development of renal dysfunction, we further
examined the relationship between genetic variants in PRKCB and baseline
renal function in an independent cross-sectional cohort of Chinese
patients with type 2 diabetes recruited in Shanghai (eTable 3). We did
not detect association between variants of PRKCB and baseline eGFR
(p=0.66-0.82) or CKD (defined as eGFR<60 ml/min per 1.73 m2,
n=195)(p=0.27-0.53) after adjustment for age, sex and BMI. This is
similar to an analysis based on analysis of all cross-sectional data from
baseline eGFR in our genotyped patients (data not shown).

Functional Annotation of the Implicated Genetic Variants in PRKCB

[0076] We performed extensive bioinformatics analyses on the functional
significance of the genetic variants identified. One of these variants,
rs3760106, is known to lie in potential binding sites for the
transcription factor Sp1 suggesting possible interaction between DNA and
binding proteins23. Further bioinformatics analysis (summarized in
eTable 4) suggest that rs3760106, rs2575390 and rs3900007 all lie within
the conserved sequence of the PRKCB promoter, whist rs2575390 and
rs3900007 both lie within histone modification sites (H3K27me3) and may
thereby alter transcriptional activity of the gene. The rs7404928 and
rs4787733 variants likewise lie within DNA regions with predicted
functional significance which may alter the binding of regulatory factors
and thereby lead to altered gene transcription (eTable 4).

Comment

[0077] In this disclosure of patients with type 2 diabetes followed up for
8 years, we found that genetic variants of the PRKCB gene predicted
development of incident ESRD independent of other known risk factors,
with joint effects amongst the risk-conferring alleles. These
associations persist despite correction for retinopathy, albuminuria,
renal function, risk factor control and use of medications including ACEI
at baseline. The strength of our study includes the relatively long
duration of follow-up, detailed documentation of clinical parameters and
drug use, as well as the use of ESRD as outcome measure for DKD. Our
consistent results thus suggest that genetic variation in the PRKCB gene
is an important determinant for the risk of developing DKD in patients
with type 2 diabetes, such as Chinese patients.

[0078] Our novel finding of an additive effect of increasing number of
PRKCB variants in predicting diabetic kidney disease is unexpected,
especially given that several of the variants were in non-coding regions.
This may reflect effects of these variants on binding of other
transcription factors, or other epigenetic effects, as predicted by our
bioinformatics analyses. In this disclosure, our results indicate that
the risk associations of the PRKCB polymorphisms with ESRD were
independent of glycaemic control suggesting that other non-glycemic
pathways may be implicated.

[0079] Taken together, our findings suggest that the risk association of
renal disease with PRKCB may be mediated by interacting pathways
including, but not limited to hyperglycaemia, and increased oxidative
stress to cause tubular damage and interstitial fibrosis.

[0080] Our study was designed to examine the association between variants
of PRKCB and development of renal dysfunction. Furthermore, given our
biological understanding of the interaction between hyperglycaemia and
activation of protein kinase C-β, one may expect the impact of
variants of PRKCB to be specific for subjects with diabetes.

[0081] Given the important role of PKC-β in the pathogenesis of DKD
and other diabetic vascular complications, a selective inhibitor of
PKC-β, Ruboxistaurin (RBX), has been developed. This drug normalized
haemodynamic changes, ECM accumulation, and histological features of
glomerular damage associated with diabetes in several animal
models18 19 20. In a Phase 2 clinical trial involving 123 type 2
diabetic patients with DKD treated optimally with ARB or ACEI, treatment
with RBX further attenuated urinary TGF-β levels, decreased rate of
loss of glomerular filtration rate and reduced proteinuria22 57.
Preliminary analysis of markers of renal disease in patients with
diabetic peripheral neuropathy participating in clinical trials of RBX
also suggests improved renal parameters during the study period58
59. Given the phenotypic and genetic heterogeneity of complex diseases
such as DKD, it would be of interest to examine the interaction between
PRKCB genotype and clinical response to RBX, as recently demonstrated in
the case for clinical response to ARB in subjects with the ACE
insertion/deletion polymorphism60.

[0082] In summary, we found that genetic variants of the PRKCB gene
predicted incident ESRD, independent of confounders notably, albuminuria,
glycaemia, retinopathy and other risk factor control.